One of the major next genomic goals is the functional annotation of the over tens of thousands of protein- encoding, non-coding RNA and micro-RNA genes of unknown in vivo function. We have developed intronic- based mutagenesis for the zebrafish as an method for effective and regulated loss-of-function approach for this model vertebrate. We have developed gene-breaking and translational gene trap vectors as insertional mutagens as new approaches for the genome-wide assignment of function using forward (phenotype- based), reverse (sequence-based) and expression-based genetic approaches in this model vertebrate. We will achieve our goal to functionally annotate unknown genes in zebrafish by accomplishing the following specific aims: Develop and test GBTs specifically designed for forward genetic screens to isolate novel genes required for zebrafish development and substance addiction. We will develop and test our 3'gene trap vector as an insertional mutagen suitable for forward genetic screening in zebrafish. Biological assays for the identification of visibly apparent defects in embryogenesis, vascular development, and a novel behavioral assay for non-visible effects of mutations on the addiction process will be employed to measure the functional mutagenicity of this vector. Develop and test GBTs specifically designed for expression-based genetic screens for the isolation of genes with restricted expression patterns that are required for zebrafish development and substance addiction. We will develop and test our red fluorescent protein (RFP)-based translational gene trap vector for the isolation of genes with tissue-specific expression patterns. . Develop and test gene-break transposons for sequence-based genetics in zebrafish. We will generate a sequence-based database of insertional alleles in genes of high interest from GBTs. These lines will be catalogued in silico and stored as cryopreserved sperm for subsequent use by the zebrafish community for sequence-based searches, regulated mutagenesis studies, and as the starting point for null allele generation. The biological reagents developed over the course of this project will meet the goals of PAR-05-080 by providing the zebrafish community with tools that will allow the functional annotation of hundreds of protein- encoding and ncRNA genes necessary for development and growth of zebrafish as well as novel genetic loci involved with vascular biology, substance addiction and vertebrate development.